Abstract 3993: The BH3-only Protein Bnip3 Mediates Mitochondrial Autophagy via Recruitment of the Ubiquitin Ligase Parkin
Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death in the heart. Bnip3 has also been shown to induce autophagy in many cells. Here, we investigated whether Bnip3 induced autophagy in primary adult cardiac myocytes by utilizing adenoviral infections of cells coupled with fluorescence deconvolution microscopy. Imaging of myocytes infected with adenoviruses expressing GFP-LC3 and Bnip3 or beta-gal were used to monitor formation of autophagosomes in cells. Overexpression of Bnip3 induced rapid increase in autophagy in adult cardiac myocytes (94.0±4.1%; n=3; p<0.05). Moreover, staining of myocytes with COX IV to label mitochondria showed extensive co-localization between mitochondria and autophagosomes after 24 h of Bnip3 overexpression. Interestingly, by 48 h many of the myocytes had lost more than half of its mitochondria, suggesting that mitochondrial biogenesis was inhibited. However, these myocytes were still viable and displayed a rodshaped morphology. Moreover, BH3-only proteins such as Bnip3 have been shown to increase production of reactive oxygen species (ROS), elevate intracellular Ca2+, and induce mitochondrial permeability transition pore (mPTP) opening, all of which have been reported to stimulate autophagy. However, Bnip3-mediated autophagy (92±3.4%) was not attenuated in the presence of the ROS scavenger N-acetyl cysteine (90.3±4.4%), the Ca2+ chelator BAPTA-AM (91.4±2.5%), or the mPTP inhibitor cyclosporine A (90.0±5.0%) (n=3, p<0.05). Cyclophilin D (cycD) is an essential component of the mPTP but Bnip3 still induced autophagy in fibroblasts isolated from cycD−/− mice (96.4±1.4% vs. 86.5±6.6% in wt, n=3). Moreover, Parkin is an ubiquitin ligase that is selectively recruited to dysfunctional mitochondria which are then removed by autophagosomes. Bnip3 induced translocation of Parkin to mitochondria in myocytes and Parkin positive mitochondria co-localized with autophagosomes. However, Parkin did not translocate to mitochondria in Bax/Bak deficient mouse embryonic fibroblasts overexpressing Bnip3. This suggests that Bax/Bak-mediated mitochondrial dysfunction by Bnip3 induces translocation of Parkin and removal of these mitochondria by autophagosomes.